Wireless swnt sensor integrated with microfluidic system for various liquid sensing applications

ABSTRACT

Sensors based on single-walled carbon nanotubes (SWNT) are integrated into a microfluidic system outfitted with data processing and wireless transmission capability. The sensors combine the sensitivity, specificity, and miniature size of SWNT-based nanosensors with the flexible fluid handling power of microfluidic “lab on a chip” analytical systems. Methods of integrating the SWNT-based sensor into a microfluidic system are compatible with the delicate nature of the SWNT sensor elements. The sensor devices are capable of continuously and autonomously monitoring and analyzing liquid samples in remote locations, and are applicable to real time water quality monitoring and monitoring of fluids in living systems and environments. The sensor devices and fabrication methods of the invention constitute a platform technology, because the devices can be designed to specifically detect a large number of distinct chemical agents based on the functionalization of the SWNT. The sensors can be combined into a multiplex format that detects desired combinations of chemical agents simultaneously.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No.61/584,857 filed Jan. 10, 2012 and entitled “Wireless SWNT SensorIntegrated With Microfluidic System for Various Liquid SensingApplications”, the whole of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was developed with financial support from Grant No.0731102 from the National Science Foundation. The U.S. Government hascertain rights in the invention.

BACKGROUND

Pollutants in water have significant impact on human health and thenatural environment. Environmental contamination, such as high levels ofnutrients, industrial wastes, toxic chemicals, and algal blooms can leadto mass mortality in fish and seabirds and may possibly result indisease outbreaks. Conventional water quality evaluation is typicallyconducted by on-site sampling followed by transport to a laboratory fortesting, or on-site data collection. Such procedures are costly, timeconsuming, and require skilled operators. Further, the test results canonly indicate the quality of water at the specific time and location ofsampling. Water quality preferably should be monitored in a fast andefficient manner and in real time which allows the responses to be usedto adequately address the sources of water contamination as quickly aspossible. Recent developments have shown a trend toward continuous datacollection using in situ detectors [1].

Carbon nanotubes have a high aspect ratio, large surface area, andunique electrical properties that offer great potential in chemical andbiological sensing applications [2, 3]. Nanotube based sensors exhibitfast response (less than 5 seconds in response to pH buffers [4]), highsensitivity (down to 20 ppb in response to dimethyl methylphosphonate inwater [5]) and are miniature in size. As an essentially one-dimensionalnanomaterial composed of a single atomic layer, single-walled carbonnanotubes (SWNT) are extremely sensitive to chemical and environmentalconditions, and the conductance of SWNTs can change dramatically whenexposed to a low concentration of ions or molecules in liquid. VariousSWNT sensors have been developed and utilized for liquid analysis,including sensors for pH value [4], heavy metal ions [6], toxic organics[5], bacteria [7], and viruses [8].

Microfluidics technology is being increasingly applied in chemical,biological and medical diagnostics of solution-based samples.Microfluidics offers numerous attractive features, such as the abilityto use small amounts of samples or reagents; to carry out sampleseparations and detections with high resolution and sensitivity; tosignificantly reduce the cost per analysis; to replace batch analysiswith continuous flow analysis; and to reduce the footprint of analyticaldevices [9]. Microfluidic systems are able to manipulate and examinesamples containing a single cell or a single molecule [10], which isespecially important for bioanalysis. Use of droplet fluidics is a newtrend in microfluidics systems, which includes the control of thedroplet volume, chemical concentrations within the droplet, and sortingof droplets based on flow pattern [11].

Integration of an SWNT sensor with a microfluidic system would enablethe development of a lab-on-a-chip device that can perform water qualitymonitoring and other types of analysis of liquid samples. Such a chipwould replace many types of measurement that are normally performedmanually in a lab using bulky equipment. With a suitable design, themicrofluidic system can carry out sample preparation steps, includingfiltering and separating various components in a liquid sample, and thenguide the solution to a nanosensor array for analysis.

In order to develop a highly sensitive and autonomous microdevice forreal-time in situ water quality monitoring, the SWNT sensors have to beintegrated with the microfluidic system, e.g., on a single chip.Permanent bonding of a microfluidic channel onto a silicon substraterequires treatment with an oxygen plasma. However, such a plasmatreatment of a carbon nanotube-based device would damage the nanotubes.Fu and colleagues integrated a SWNT sensor with a microfluidic channelby covering the SWNT with a continuous metal layer which protected thenanotubes underneath during exposure to oxygen plasma [12]. Afterbonding the SWNT device with a microfluidic channel made ofpolydimethylsiloxane (PDMS), an etching solution was introduced throughthe channel to remove the metal mask layer covering the SWNT, so thatthe SWNT inside the microfluidic channel could be used for sensingapplications [12]. However, this type of integration process couldintroduce contamination onto the SWNT from the etching solution and thesubstances generated by the chemical reactions. Bourlon and colleagueshave fabricated a flow and ionic sensor using a nanotube transistorcovered with a PDMS channel without using an oxygen plasma treatment[13]; however, the microfluidic channel was not well sealed on thedevice which resulted in leakage of solution. Thus, there remains a needto develop procedures and devices that incorporate SWNT intomicrofluidic channels without compromising the integrity of the SWNT orthe fluid handling of the microfluidics system.

SUMMARY OF THE INVENTION

The present invention integrates the sensitivity, specificity, andminiature size of SWNT-based sensors with the flexible fluid handlingpower of microfluidic “lab on a chip” analytical systems. The furtheraddition of data analysis and wireless transmission capabilities resultsin a nanosensor device capable of either continuous or intermittentsampling and analysis of liquid samples, especially in remote or poorlyaccessible locations. The sensor device is directly applicable to waterquality monitoring and bioanalytical monitoring in living systems. Themethods of fabricating microfluidic embodiments of the sensor device arecompatible with the delicate nature of the sensor elements.

One aspect of the invention is a sensor device for the detection of achemical agent in a liquid sample. The device includes a substrate, apatterned conductive layer deposited on a surface of the substrate andforming first and second microelectrodes with a gap between themicroelectrodes, a nanosensor connected to the microelectrodes andtraversing the gap, and a microfluidic channel that contains thenanosensor within its lumen. The nanosensor includes one or moresingle-walled carbon nanotubes (SWNT) that have been assembled acrossthe microelectrodes using dielectrophoresis. In some embodiments, theSWNT have been functionalized so as to provide a change in an electricalproperty of the SWNT in response to a particular chemical agent oranalyte. In some embodiments, the microfluidic channel defines a fluidpathway that connects the lumen containing the nanosensor with an inletport and an outlet port, through which the liquid sample for analysis isadded to and removed from the device, respectively. The ports can be onany surface of the device, including the substrate, the conductivelayer, or a housing for the device. Optionally, the microelectrodes, orportions of the conductive layer connected with them, are connected to adetection circuit formed by part of the conductive layer or mounted onthe substrate. The detection circuit applies a voltage across the SWNTsensor element through the microelectrodes, detects a change in anelectrical property of the SWNT in response to binding of the chemicalagent to the SWNT or a functional group on the SWNT. The change inelectrical property is preferably a change in resistance or capacitanceof the SWNT. The detection circuit then produces an output signal, suchas a voltage, that is related to the presence and/or amount orconcentration of the chemical agent in the test sample.

Another aspect of the invention is a method of fabricating the sensordevice described above. The method includes the steps of: (a) depositinga conductive layer onto an insulating or semiconducting substrate,whereby the conductive layer forms a pattern that includes first andsecond microelectrodes and having a gap between the first and secondmicroelectrodes; (b) forming a microfluidic channel that covers aportion of the substrate, encloses the gap, and is fluidically connectedon one side of the gap to an inlet port and on another side of the gapto an outlet port; (c) flowing an aqueous suspension of SWNT through theinlet port to fill the enclosed gap with the aqueous suspension; (d)applying an AC voltage between the first and second microelectrodes,whereby SWNT are dielectrophoretically assembled across the gap and forman electrical connection at one end of the SWNT with the firstmicroelectrode and at another end of the SWNT with the secondmicroelectrode; and (e) removing the aqueous suspension of SWNT from themicrofluidic channel.

Yet another aspect of the invention is another method of fabricating theabove-described sensor device. The method includes the steps of: (a)depositing a conductive layer onto an insulating or semiconductingsubstrate, whereby the conductive layer forms a pattern comprising firstand second microelectrodes and having a gap between the first and secondmicroelectrodes; (b) depositing an aqueous suspension of SWNT onto thesubstrate so as to cover the gap between the first and secondmicroelectrodes with the aqueous suspension; (c) applying an AC voltagebetween the first and second microelectrodes, whereby SWNT aredielectrophoretically assembled across the gap and form an electricalconnection at one end of the SWNT with the first microelectrode and atanother end of the SWNT with the second microelectrode; (d) removingsaid aqueous suspension of SWNT from the substrate; (e) forming aparylene shadow mask covering the assembled SWNT; (f) plasma treatingthe substrate; (g) removing the shadow mask; and (h) bonding amicrofluidic channel to the substrate that encloses the gap and theassembled SWNT, and is fluidically connected on one side of the gap toan inlet port and on another side of the gap to an outlet port.

Still another aspect of the invention is a method of detecting orquantifying a chemical agent in a liquid sample. The method includes thesteps of: (a) providing the above-described sensor device; (b) flowingthe liquid sample into the inlet port of the sensor device so as to fillthe enclosed gap; and (c) observing an output signal from the detectioncircuit of the sensor device. The observed output signal indicates thepresence or absence of, and/or the amount or concentration of thechemical agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a) shows a schematic illustration of an SWNT sensor inside amicrofluidic channel for continuous flow-through monitoring of a liquidsample. FIG. 1( b) shows a view of the sensor with the microfluidicchannel removed.

FIG. 2 shows an embodiment of a fabrication process for integrating SWNTsensors inside a microfluidic channel. FIG. 2( a) shows theconfiguration of Cr/Au microelectrodes that were fabricated byphotolithography on a SiO₂ substrate. FIG. 2( b) shows a microfluidicchannel that has been bonded onto the Si chip from FIG. 2( a). FIG. 2(c) illustrates how a dielectrophoretic process was utilized to assembleSWNT at appropriate location and with appropriate alignment inside themicrofluidic channel. SWNT solution was transported to the electrodesinside the channel by the microfluidic system and an AC electric fieldwas applied to the electrode pads outside the channel. FIG. 2( d) showsthe fabricated SWNT sensors integrated within the final microfluidicsystem.

FIG. 3 shows scanning electron microscope (SEM) micrographs of SWNTassembled on Au microelectrodes. In FIG. 3( a), nanotube bundles arevisible which are immobilized on the electrodes after assembly. FIG. 3(b) shows SWNT assembled on a microelectrode after exposure to oxygenplasma protected with a parylene mask. The coverage of SWNT with aparylene layer successfully protected nanotubes from being damaged bythe oxygen plasma.

FIG. 4 shows the I-V characteristic of a SWNT sensor integrated inside amicrofluidic channel.

FIG. 5( a) shows a photographs of the testing board and the sensor arraywith a 3 μm gap between the electrodes enlarged in the insertmicrograph. FIG. 5( b) shows a wireless transmission platform. FIG. 5(c) is a schematic illustration of the testing circuitry for the SWNTsensor. The insert in FIG. 5( a) illustrates the pattern of the chip andthe structure of the Au electrodes. FIGS. 5( d)-5(f) show variousconfigurations of the sensor device with different modes of outputtingthe results of the measurements.

FIG. 6 shows the resistance of an SWNT sensor after exposure todeionized water and different pH buffers. The sensor was rinsed withdeionized water and dried in air before introducing each different pHsolution. The resistance of the SWNT dropped back to essentially thesame value after each cycle of rinsing and drying, which indicated thatthe nanotubes were stable during pH sensing.

FIG. 7 shows the percentage change in resistance of carbon nanotubesmeasured in buffer solutions from pH 5 to pH 8 and from pH 8 to pH 5.The forward test (from pH 5 to pH 8) is plotted with round dots and thebackward test (from pH 8 to pH 5) is plotted with square dots.

FIG. 8 shows the resistance of a SWNT sensor inside a microfluidicchannel when exposed to aqueous solutions with different pH values. Thebuffer solution inside the microfluidic channel was continuously changedfrom pH 8 to pH 5 via a syringe. This test mimics real-time pHmonitoring of a liquid sample.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed methods for integrating sensors based onsingle-walled carbon nanotubes (SWNT) into a microfluidic system, andoptionally outfitting the integrated device with data processing andwireless transmission capability. The sensors combine the sensitivity,specificity, and miniature size of SWNT-based nanosensors with theflexible fluid handling power of microfluidic “lab on a chip” analyticalsystems. The methods developed can be used to fabricate new sensordevices by constructing a microfluidic system using techniques that arecompatible with the delicate nature of the SWNT sensor elements. Themicrofluidic mechanism enables the sensor devices to continuously andautonomously monitor liquid samples for the presence or absence and/orconcentration of desired chemical agents. The addition of wireless datatransmission extends the capability of the sensor devices to real timeremote sensing operations, sensing in remote locations, and sensing ofselected fluid-containing spaces in living organisms. The sensor devicesof the invention are well suited to continuous monitoring of waterquality in the environment as well as in industrial and home settings.The sensor devices and fabrication methods of the invention provide aplatform technology. Their specificity for detecting different chemicalagents can be varied according to the functionalization of the SWNT usedas the sensor element. Different sensors also can be combined in amultiplex format, so that desired combinations of chemical agents can bedetected or analyzed simultaneously.

A schematic illustration of one embodiment of a nanotube-microfluidicsensor is presented in FIG. 1( a). The overall configuration of thesensor includes a substrate having a patterned conductive layerdeposited on a surface of the substrate, and an attached microfluidicchannel that encloses the sensor element on the substrate. The substrateis made from or contains an electrically insulating (non-conducting) orsemiconducting material such as silicon, silicon oxide, glass, or apolymer such as a non-conducting organic polymer, or any combinationthereof. The substrate is typically flat and rigid, but its shape andthickness can be chosen according to use. The substrate can also beflexible and either opaque or transparent. It can also be made of abiocompatible material. The surface area of the substrate can beselected by the user's requirements, but it can be quite small, evensmall enough to fit in a blood vessel of a mammal such as a human.

In FIG. 1( b), microfluidic channel housing 200 is shown detached fromthe lower portion of sensor device 100. The microfluidic channel can befabricated in any material known for use in microfluidics, such aspolydimethylsiloxane (PDMS), glass, silicon, a polymer, or anycombination thereof. The channel housing material is preferablyhydrophilic, so that water and aqueous solutions can readily betransported through it with a minimum of resistance. The channel can besilanized or otherwise coated to reduce binding or adsorption of theanalyte or interfering substances.

Substrate 10 is overlaid with a conductive layer which forms firstmicroelectrode 21 and second microelectrode 22; the microelectrodes arelinked via conductive pathways to contact pads 20 and 26, respectively,through which electrical contact is made with a detection circuit (notshown). The conductive layer can be formed by depositing a conductivematerial, e.g., by sputtering, chemical vapor deposition, or physicalvapor deposition, and patterned using a lithography process. Suitableconductive materials include Au, Ag, Al, Cr, Cu, conductive polymers,metal particle composites, metal-polymer particle composites, nanotubes,and combinations thereof. The microelectrodes are each in electricalcontact with one end of nanosensor 30, which contains one or moredielectrophoretically assembled SWNT. The microelectrodes are disposedon the substrate leaving a non-conductive gap between them. The gap canbe filled with air, vacuum, or a non-conducting material. The gap ispreferably on the order of about 1 μm to about 10 μm in length. The sizeof the gap is selected so as to be conveniently bridged by an assemblyof one or more SWNT formed by a dielectric assembly process whichinvolves establishing an AC voltage between the microelectrodes in thepresence of an aqueous suspension of individual dispersed SWNT.Optionally, an AC voltage source can be combined with a DC offsetvoltage so that a combination of dielectrophoresis and electrophoresisis used for assembly. Methods for dielectric assembly of SWNT are wellknown in the art.

The SWNT used for dielectric assembly can be metallic SWNT,semiconducting SWNT, nonconducting or insulating SWNT (which can beadded to adjust the electrical properties of the nanosensor, e.g., toadjust based on size of the sensor or type of functionalization),functionalized SWNT, non-functionalized SWNT, or any combinationthereof. Functionalization of SWNT is well known in the art. Forexample, a glucose sensor can be fabricated using SWNT functionalized bycoating them with the enzyme glucose oxidase. A sensor for a specificcell type, such as a bacterial species or strain, or a virus species orstrain, can be fabricated using SWNT functionalized by coating with anantibody specific for the cell, bacterium, or virus. A sensor for aspecific organic compound, such as a pollutant or toxin, can befabricated using SWNT functionalized by coating with an antibody or acompound that binds with or reacts with the target compound.Non-functionalized SWNT are sensitive to H. SWNT can be assembled ontothe first and second microelectrodes either in a pre-functionalizedstate, or they can be assembled in an already functionalized state.Pre-functionalized SWNT can be SWNT that have appropriate chemicalsubstituents attached to the SWNT, such as —COOH, —NH₂, or —SH groups,and after assembly the specific binding or reacting agent (e.g.,antibody) can be coupled to the SWNT, which are then functionalized insitu by introducing the appropriate reactants and reaction conditionsusing the microfluidic system.

Microfluidic channel 40 is a fluid passageway having inlet port 45 andoutlet port 47 accessible from the side not attached to the substrate,and having an open space on the underside of the channel housing inposition to enclose gap 25 between the microelectrodes and nanosensorelement 30. The open space of the channel is fluidically connected withthe inlet port on one side of the space and with the outlet port on theother side of the space, such that sample liquid can flow in through theinlet port, across the nanosensor element, and then out through theoutlet port. The terms “inlet port” and “outlet port” do not requirethat the port have an opening at the surface of the microfluidicshousing or the device housing, although it may. The port can also be aconnection to another fluid channel or pathway within the device.Optionally, the fluid can be directed not to inlet and outlet ports butto one or more additional microfluidic channels, reservoirs, pumps,vacuum supply channels, reaction chambers, filters, or processingchambers on the same substrate or on an attached or separatemicrofluidic module or chip. Fluid can be moved through the microfluidicchannel by any means known in the art of microfluidics, including theuse if a pump (e.g., a piezoelectric device), capillary action alone, orvacuum applied through a port on the device attached to a vacuum source.Optionally, tubing can be attached to the inlet and/or outlet ports; theother end of the tubing can be placed into, for example, a sample fluidreservoir, a natural body of water or sampling fluid space, wastereceptacle, syringe, or pump.

It should be noted that the sensor device can be configured as a singlesensor, as a compound sensor having two or more iterations of a singletype of sensor (either sharing common fluid sample pathways or eachhaving its own fluid sample pathway), or as a multiplex sensor havingtwo or more different types of sensor (e.g., each using differentlyfunctionalized SWNT and therefore sensitive to a different chemicalagent).

The invention contemplates two different fabrication processes withwhich to integrate SWNT nanosensors into a microfluidic system. Bothprocesses are efficient and result in high yield. The first approach isschematically illustrated in FIG. 2. Microelectrodes with a gap of 3 μmwere fabricated by photolithography and metal deposition (Cr/Au, 20nm/150 nm) on an oxidized silicon wafer (FIG. 2( a)). A PDMS slabembossed with a microfluidic channel (length/width/height: 8 mm/2 mm/150μm) was cast using SU-8 replica molding which was created byphotolithography [14]. The silicon chip with the microelectrodes and themicrofluidic channel was exposed to an oxygen plasma (Plasma-Therm 790)for 30 seconds and then placed in contact with a hot plate at atemperature of 150° C. for 15 min (FIG. 2( b)). This process generated apermanent seal between the PDMS channel and the silicon chip [15]. Twoholes were punched with a syringe needle on the microfluidic channel andTygon tubes (Small Parts, Inc.) were inserted into the holes. The SWNTwere next assembled between Au electrodes by a low temperature,dielectrophoresis (DEP) process inside the microfluidic channel (FIG. 2(c)). In this process, commercially available SWNT (diameter: 1-2 nm andlength: 2-5 μm) in aqueous suspension at a concentration of 40 mg/L wereinjected into the microfluidic channel using a syringe. An AC field of 5Vpeak-to-peak and 300 KHz was applied between the two electrodes for 1min to assemble the nanotubes. After assembly, the remaining SWNTsolution inside the microchannel was removed by injecting air into thechannel. During dielectrophoretic assembly, carbon nanotubes wereimmobilized on Au electrodes by Van der Waals forces (FIG. 2( d)). I-Vmeasurements confirmed the assembly of SWNT inside the microfluidicsystem.

In the second approach, the nanotubes were first assembled between themicroelectrodes using the DEP process described above, which wasperformed prior to adding the microfluidic channel. While conducting theoxygen plasma treatment, a 10 μm thick parylene mask (fabricatedaccording to Selvarasah et. al. [16]) was placed onto the chip, whichprevented exposure of SWNT to the oxygen plasma required to bond themicrofluidic channel. SEM images of SWNT assembled on microelectrodesbefore and after plasma treatment protected with the parylene mask werecompared in FIGS. 3( a) and 3(b), respectively. Carbon nanotubes formeda network between the electrode (the bottom round shaped pad) and thesubstrate and the network was comprised of several SWNT bundles (FIG. 3(a)). No damage was observed to the SWNTs after plasma treatment in FIG.3( b) with the protection of 10 μm parylene film. The shadow mask can bepeeled off using either mechanical or chemical means. Mechanical means(pulling away the mask) are preferred, since chemical removal can leavean interfering chemical residue from the mask. After peeling off theshadow mask, the SWNT device was bonded with a PDMS microfluidic channelon a 150° C. hot plate for 15 min. The I-V characteristics of SWNTbonded with the microfluidic channel was measured and is illustrated inFIG. 4. The resistance of the SWNT decreased from 14 KΩ to 11 KΩ afterbonding the microfluidic channel on a 150° C. hot plate. This waspossibly due to reduction of the contact resistance between assembledSWNTs and metal electrodes caused by the thermal treatment [17].

The SWNT sensor integrated with microfluidic channel was next wirebonded onto a ceramic chip holder. As shown in FIG. 5( a), thefabricated device was mounted by means of the chip holder onto acircuitry board for signal conditioning (the corresponding circuitry isdescribed in FIG. 5( c)). The change in resistance of the nanosensorcaused by the presence of analytes (in this case, H⁺) in the liquidsample was converted into a change in output voltage and transmitted,where the resistance of carbon nanotubes (Rswnt) was related to theoutput voltage by Equation 1 and Rswnt can be calculated by equation 2:

Rswnt/(Rswnt+R)=Vout/Vcc  (1)

Rswnt=R×Vout/(Vcc−Vout)  (2)

Rswnt is the resistance of the SWNT; R is an external reference resistor(10 KΩ); Vout is the output voltage and Vcc is the voltage of the powersupply. A wireless sensing platform (Waspmote) was used to acquire theoutput voltage and wirelessly transmit the data to a remote receiver (asshown in FIG. 5( b)), which could be directly connected to a computer.

A sensor device according to the present invention can be configured ina number of different ways to suit the needs of the user. For example,the device can be incorporated into another device, such as ananalytical device, a “lab on a chip” system, or it can be used as astand-alone sensor. The sensor device can include only the nanosensorwithout any in-built detection circuitry, memory, or data processingcircuitry, or it can optionally be configured to include, either on thesame substrate (i.e., chip) or on an attached substrate, any one or moreof the following optional components, or other components: a detectioncircuit, a data processing circuit, a wireless transmission module, anantenna, a microprocessor, a memory chip or hard drive, and a battery.The sensor device can be incorporated into a housing and be outfittedwith a display, such as an analog or digital display to output a valueof an electrical property of the sensor or a calculated or computedvalue of an amount or concentration of a chemical agent or analyte. FIG.5( d) shows an embodiment (300) that includes sensor device 100 attachedthrough one or more electrical leads 125 to detection circuit 255, whichin turn has one or more output leads 255 that convey one or more outputvalues off of the device. Input and output lines 110 and 120 provide afluid pathway onto and off of the device for the sample liquid. FIG. 5(e) shows an embodiment (350) that further includes transmitter module360, which transmits a signal, such as a radio frequency signal, fromthe device through antenna 370. FIG. 5( f) shows another embodiment(400) that includes data processing module 380 interposed betweendetection circuit 250 and transmitter 360. The data processing circuitrymay also be combined with the detection circuit and/or the transmittercircuit into a single electrical module. Optionally, the data processingand/or detection circuits can be programmed through direct contacts,buttons or a touch screen display on the device, or remotely through areceiver module or combined transmitter/receiver module in conjunctionwith a cell phone, tablet computer, laptop computer, or remote computersystem.

The sensor device can be supplied as the device alone or as a kit, withinstructions for use, and optionally with one or more reagents, such asreagents for SWNT in situ functionalization, reagents for modifying thesample prior to detection, etc. Certain microfluidics embodiments can bepreloaded with one or more reagents when supplied as a kit. The devicecan also be provided with microfluidics channel attached but lacking theSWNT sensor, which can be added by performing DEP with the user's ownfunctionalized SWNT.

The pH value of water in rivers and lakes may change due to introductionof pollutants through human activities including automobile exhaust(generation of acid rain), accidental spills, agricultural run-off andsewer overflows. Infant fish and insect larvae are sensitive to low pH(acidic) aqueous environments, and extreme values of pH can be lethal tomost organisms [18]. Monitoring the pH value of water is critical toprovide an early warning to the environmental authorities. Due to theunique properties of nanomaterials, including carbon nanotubes, boronnitrite nanotubes, and grapheme, they have recently attractedconsiderable attention from researchers for the detection of pH value inwater [19-21]. However, prior to the present invention, there does notappear to have been available a microfluidic system which is adapted forcontinuous pH monitoring at remote locations.

The fabricated nanosensor system was applied to detect pH values ofaqueous solutions. The expected pH range in rivers is between 6.5 and9.0 [22]. Therefore, buffers with pH values of 5, 6, 7, and 8 wereprepared and calibrated using a commercial pH meter [Hach Inc.]. It isnoted that the only effects of ions and molecules that can be sensed bySWNT-based sensors are those acting within the Debye-length. The Debyelength in this case can be simply defined as the typical distancerequired by charged molecules surrounding the nanotubes to be sensed bythe charge carriers inside the nanotubes, and it is inverselyproportional to the square root of the ionic strength (I) in liquid[23]. In water, the Debye length is ˜0.32 I to 0.5 nm. In order tomaintain the same Debye length for SWNT in the various pH buffersolutions, the ionic strengths of the pH buffer solutions were heldconstant at 100 mM. With the same Debye length in the different pHbuffer solutions, the conductance of the SWNT was only affected by thepH values of solutions.

The output voltage of the nanosensor system during pH detection wasrecorded and converted back into the resistance of nanotubes by acomputer. In FIG. 6, the SWNT resistance was plotted versus time, andthe liquid to be tested was changed from deionized water to buffersolutions with increasing pH values, and then from buffer solutions withdecreasing pH values back to deionized water. The resistance of the SWNTsensor increased from 50.26 kΩ to 61.54 kΩ when the pH values inside themicrofluidic channel increased from 5 to 8. In addition, the forwardmeasurement (pH values from 5 to 8) exhibited an opposite trend comparedto the backward measurement (pH values from 8 to 5) which indicated thatthe response of SWNT nanosensor to pH was reversible and repeatable.Between each solution change, the SWNT sensor was rinsed with deionizedwater thoroughly and dried with compressed air. After the SWNT werewashed and dried, the initial resistance of SWNTs was essentiallyrecovered, which implied that SWNT sensor was able to maintain aconsiderable level of stability when utilized in a liquid environment.Moreover, when the changes in resistance of the SWNT sensor duringexposure to different pH buffers were calculated and plotted in FIG. 7,the response of the sensor was essentially linearly proportional to thepH value of solutions inside the microfluidic channel.

The SWNT nanosensor integrated into a microfluidic system was testedduring real-time pH monitoring under continuous sample liquid flowconditions. The resistance of the SWNT sensor was recorded as a functionof time, during which the solution inside the microfluidic channel waschanged to different pH buffers. As shown in FIG. 8, the resistance ofthe SWNT increased from 5.55 kΩ in deionized water to 7.27 kΩ afterexposure to pH 8 buffer solution (the change in resistance was about31%). When pH 7 buffer was introduced into the channel, the resistanceof the nanotubes gradually decreased to 6.85 kΩ. As the pH values of thesolutions inside the channel decreased, the resistance of SWNT sensordecreased immediately and reached a stable value within a few seconds.The response of the microfluidics-integrated nanosensor to continuous pHchanges showed the same trend as the data plotted in FIG. 6, obtainedunder fluid static conditions. In addition, this real-time measurementshowed that the response time of the SWNT sensor to the pH value of theaqueous solution was less than 10 seconds. All the measurementsdescribed above were repeated three times with five different samples,and the results were confirmed.

The integration of a carbon nanotube-based sensor with a microfluidicsystem according to the present invention provides for continuous andremote monitoring of liquid samples, such as for water qualitymonitoring and for monitoring of fluids in biological systems includingcell culture media and various fluid spaces in the bodies of livinganimals or body fluid samples taken from living animals. The SWNT-basedsensor device has many superior properties compared to conventionaldetection equipment, and it has high sensitivity, fast response time,and is well suited for miniaturization of analytical devices. With theaddition of one or more microfluidic sub-systems customized for liquidsampling, sample preparation, sample purification, or carrying outchemical pre-treatment of the sample before testing, the SWNT-basedsensor can be utilized for autonomous monitoring in remote environments,such as for water quality monitoring.

The invention includes a method of detecting or quantifying a chemicalagent or analyte in a liquid sample. Using a sensor device as describedabove, a liquid sample is allowed to flow into the microfluidic channeland to fill the enclosed gap (sensor chamber) of the device. The outputsignal, or a change in the output signal, is then observed using thedetection circuit, either by directly probing the circuit, or byreceiving an output value from a display on the device, or by receivinga value from the data processing module either directly, throughdisplay, or remotely via the wireless transmission system. The observedsignal or change in signal is indicative of the presence or absence, orof the concentration or amount, of the chemical agent or analyte. Ifdesired, the output signal can be compared to a calibration made withthe same sensor device or a similar device in order to more accuratelyanalyze the presence and/or concentration of the agent. Sampling andmonitoring of results can be performed either continuously orintermittently, such as at predetermined times or intervals, as desired.As part of the sampling process, the sample liquid optionally can beseparated, filtered, purified, or chemically modified using themicrofluidics system.

One challenge in the integration of nanosensors and microfluidic systemsis that the plasma treatment required for PDMS bonding would ordinarilydamage the carbon nanotubes on the microdevice. In order to solve thisproblem, the inventors have developed two approaches by which anSWNT-based nanosensor can be fabricated inside of a microfluidic channelwithout damage to the sensing elements. First, the microfluidic channelcan be bonded onto a substrate, such as a silicon substrate, and thenthe SWNT can be assembled via a dielectrophoretic process carried outinside the already fabricated microfluidic channel. Second, the SWNT canbe assembled on the substrate and protected by a shadow mask, such as aparylene mask, during plasma treatment, after which the microfluidicchannel is bonded onto the device, enclosing the SWNT sensor element.The SWNT-based microfluidic sensor was tested under continuous flowconditions and found to produce a linear relationship between the changein resistance of the nanosensor and the concentration of a chemicalagent in the test liquid (e.g., H⁺ concentration in buffered aqueoussolutions). The sensing responses to analyte were reversible andrepeatable. By connecting the sensor with a signal processing board anda wireless transmission unit, the SWNT-based sensor was shown to befunctional as a remotely located independent sensing unit with a smallsize. This embodiment of the invention can be placed in rivers andlakes, and in various water transportation systems, such as householddrains and pipes for real time water quality monitoring.

As used in any claim herein, “consisting essentially of” does notexclude materials or steps whose effects are immaterial with respect tothe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, can beexchanged with “consisting essentially of” or “consisting of” todescribe alternative embodiments.

While the present invention has been described in conjunction withcertain preferred embodiments, one of ordinary skill, after reading theforegoing specification, will be able to effect various changes,substitutions of equivalents, and other alterations to the compositionsand methods set forth herein. All publications cited herein are whollyincorporated by reference.

REFERENCES

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What is claimed is:
 1. A sensor device for the detection of a chemicalagent in a liquid sample, the device comprising: an electricallyinsulating or semiconducting substrate; a conductive layer deposited ona surface of the substrate, the conductive layer comprising first andsecond microelectrodes and having a gap between the first and secondmicroelectrodes; a microfluidic channel for the passage of said liquidsample, the channel enclosing said gap and fluidically connected on oneside of the gap to an inlet port and on another side of the gap to anoutlet port; a nanosensor comprising one or more single-walled carbonnanotubes (SWNT) traversing said gap, one end of the SWNT forming anelectrical connection with the first microelectrode and the other end ofthe SWNT forming an electrical connection with the secondmicroelectrode; and a detection circuit connected to said first andsecond microelectrodes and capable of detecting a change in anelectrical property of the SWNT; wherein a change in the electricalproperty of the SWNT during passage of said liquid sample across saidgap indicates the presence and/or amount of said chemical agent in saidliquid sample.
 2. The sensor device of claim 1, wherein the electricalproperty is resistance or capacitance.
 3. The sensor device of claim 1,wherein said SWNT are functionalized such that said electrical propertychanges specifically in the presence of said chemical agent.
 4. Thesensor device of claim 1, wherein said SWNT are dielectrophoreticallyassembled.
 5. The sensor device of claim 1, comprising a plurality offirst and second microelectrode pairs, each pair having a gap betweenthe microelectrodes of the pair and having a nanosensor comprising oneor more SWNT traversing said gap, one end of the SWNT forming anelectrical connection with the first microelectrode of the pair and theother end of the SWNT forming an electrical connection with the secondmicroelectrode of the pair, and wherein each gap is enclosed by saidmicrofluidic channel.
 6. The sensor device of claim 5 that is amultiplex sensor comprising two or more different nanosensors havingsensitivity to different chemical agents.
 7. The sensor device of claim1, wherein the chemical agent is selected from the group consisting ofH⁺, metal ions, glucose, bacteria, viruses, and organic compounds. 8.The sensor device of claim 1, further comprising a data processingmodule capable of processing an output signal from said detectioncircuit and outputting a signal that provides information on thepresence and/or amount of said chemical agent.
 9. The sensor device ofclaim 1, further comprising a wireless transmitter capable oftransmitting an output signal from said detection circuit to a remotereceiver.
 10. The sensor device of claim 8, further comprising awireless transmitter capable of transmitting an output signal from saiddata processing module to a remote receiver.
 11. The sensor device ofclaim 1, wherein the substrate comprises silicon, glass, or a polymer.12. The sensor device of claim 1, wherein the conductive layer comprisesa material selected from the group consisting of Au, Ag, Al, Cr, Cu,conductive polymers, metal particle composites, metal-polymer particlecomposites, nanotubes, and combinations thereof.
 13. The sensor deviceof claim 1, wherein the microfluidic channel is formed from a materialselected from the group consisting of polydimethylsiloxane, glass,silicon, polymers, and combinations thereof.
 14. The sensor device ofclaim 1, wherein the SWNT are selected from the group consisting ofmetallic SWNT, semi-conducting SWNT, insulating SWNT, functionalizedSWNT, and mixtures thereof.
 15. The sensor device of claim 1, whereinthe gap between the first and second microelectrodes is from about 1 μmto about 10 μm in length.
 16. The sensor device of claim 1, furthercomprising one or more microfluidic modules for purifying, processing,or chemically modifying the liquid sample, and the output of said one ormore microfluidic modules is connected to said inlet port.
 17. Thesensor device of claim 1, further comprising a fluid driving mechanismfor causing fluid to flow through said microfluidic channel.
 18. Thesensor device of claim 1, further comprising one or more reservoirs forreagents that are mixed with the liquid sample by one or moremicrofluidics modules prior to contacting said liquid sample with saidnanosensor.
 19. The sensor device of claim 1, further comprising areservoir for fluid waste, wherein said reservoir is fluidicallyconnected with said outlet port.
 20. A method of fabricating a sensordevice for the detection of a chemical agent in a liquid sample, themethod comprising the steps of: (a) depositing a conductive layer ontoan insulating or semiconducting substrate, whereby the conductive layerforms a pattern comprising first and second microelectrodes and having agap between the first and second microelectrodes; (b) forming amicrofluidic channel that covers a portion of the substrate, enclosesthe gap, and is fluidically connected on one side of the gap to an inletport and on another side of the gap to an outlet port; (c) flowing anaqueous suspension of SWNT through said inlet port to fill said enclosedgap with said aqueous suspension; (d) applying an AC voltage between thefirst and second microelectrodes, whereby SWNT are dielectrophoreticallyassembled across the gap and form an electrical connection at one end ofthe SWNT with the first microelectrode and at another end of the SWNTwith the second microelectrode; and (e) removing said aqueous suspensionof SWNT from the microfluidic channel.
 21. The method of claim 20,wherein step (b) comprises: (b1) forming a microfluidic channel inpolydimethylsiloxane (PDMS) using SU-8 replica molding andphotolithography; (b2) exposing the microfluidic channel and thesubstrate to an oxygen plasma for about 30 seconds; (b3) bonding themicrofluidic channel to the substrate at about 150° C. for about 15minutes.
 22. The method of claim 20, wherein a plurality of first andsecond microelectrode pairs is formed, each pair having a gap betweenthe microelectrodes of the pair, and the gap is enclosed by themicrofluidic channel; and whereby SWNT are dielectrophoreticallyassembled across each gap and form an electrical connection at one endof the SWNT with the first microelectrode of each pair and at anotherend of the SWNT with the second microelectrode of each pair.
 23. Themethod of claim 22, wherein steps (c) through (e) are repeated for oneor more cycles of SWNT assembly; wherein at each performance of step (d)the voltage is applied between a different pair of microelectrodes;wherein for each performance of steps (c) and (d) the SWNT aredifferently functionalized, whereby a multiplex sensor is fabricated.24. The method of claim 20, further comprising: (f) attaching to thesubstrate a detection circuit connected to said first and secondmicroelectrodes and capable of detecting a change in an electricalproperty of the SWNT.
 25. The method of claim 24, further comprising:(g) attaching to the substrate a wireless transmitter capable oftransmitting an output signal from said detection circuit to a remotereceiver.
 26. The method of claim 24, further comprising: (g) attachingto the substrate a data processing module capable of processing anoutput signal from said detection circuit and outputting a signal thatprovides information on the presence and/or amount of said chemicalagent.
 27. The method of claim 26, further comprising: (h) attaching tothe substrate a wireless transmitter capable of transmitting an outputsignal from said data processing module to a remote receiver.
 28. Amethod of fabricating a sensor device for the detection of a chemicalagent in a liquid sample, the method comprising the steps of: (a)depositing a conductive layer onto an insulating or semiconductingsubstrate, whereby the conductive layer forms a pattern comprising firstand second microelectrodes and having a gap between the first and secondmicroelectrodes; (b) depositing an aqueous suspension of SWNT onto thesubstrate so as to cover the gap between the first and secondmicroelectrodes with the aqueous suspension; (c) applying an AC voltagebetween the first and second microelectrodes, whereby SWNT aredielectrophoretically assembled across the gap and form an electricalconnection at one end of the SWNT with the first microelectrode and atanother end of the SWNT with the second microelectrode; (d) removingsaid aqueous suspension of SWNT from the substrate; (e) forming aparylene shadow mask covering the assembled SWNT; (f) plasma treatingthe surface of the substrate having the assembled SWNT; (g) removing theshadow mask; and (h) bonding a microfluidic channel to the substratesuch that it encloses the gap and the assembled SWNT, and is fluidicallyconnected on one side of the gap to an inlet port and on another side ofthe gap to an outlet port.
 29. The method of claim 28, wherein step (f)comprises forming a microfluidic channel in PDMS using SU-8 replicamolding and photolithography and exposing the microfluidic channel andthe substrate to an oxygen plasma for about 30 seconds; and wherein step(h) comprises heat bonding the microfluidic channel to the substrate atabout 150° C. for about 15 minutes.
 30. The method of claim 28, whereina plurality of first and second microelectrode pairs is formed in step(a), each pair having a gap between the microelectrodes of the pair, andwherein in step (c) SWNT are dielectrophoretically assembled across eachgap and form an electrical connection at one end of the SWNT with thefirst microelectrode of each pair and at another end of the SWNT withthe second microelectrode of each pair.
 31. The method of claim 28,wherein in step (g) the shadow mask is removed mechanically.
 32. Themethod of claim 30, wherein steps (b) through (d) are repeated for oneor more cycles of SWNT assembly; wherein at each performance of step (c)the voltage is applied between a different pair of microelectrodes;wherein for each performance of steps (b) and (c) the SWNT aredifferently functionalized, whereby a multiplex sensor is fabricated.33. The method of claim 28, further comprising: (i) attaching to thesubstrate a detection circuit connected to said first and secondmicroelectrodes and capable of detecting a change in an electricalproperty of the SWNT.
 34. The method of claim 33, further comprising:(j) attaching to the substrate a wireless transmitter capable oftransmitting an output signal from said detection circuit to a remotereceiver.
 35. The method of claim 33, further comprising: (j) attachingto the substrate a data processing module capable of processing anoutput signal from said detection circuit and outputting a signal thatprovides information on the presence and/or amount of said chemicalagent.
 36. The method of claim 35, further comprising: (k) attaching tothe substrate a wireless transmitter capable of transmitting an outputsignal from said data processing module to a remote receiver.
 37. Amethod of detecting or quantifying a chemical agent in a liquid sample,the method comprising the steps of: (a) providing a sensor device ofclaim 1; (b) flowing the liquid sample into the inlet port of the sensordevice so as to fill the enclosed gap; and (c) observing an outputsignal from the detection circuit of the sensor device; wherein theobserved output signal indicates the presence or absence of, and/or theamount or concentration of, said chemical agent.
 38. The method of claim37, wherein the output signal is monitored over a period of time eithercontinuously or at selected times or time intervals.
 39. The method ofclaim 37, wherein the sensor device comprises a wireless transmitter,and step (c) comprises remotely receiving a signal from the transmitter.40. The method of claim 39, wherein the sensor device is located at anatural body of water.
 41. The method of claim 39, wherein the sensordevice is located in a plumbing system or a water purification system.42. The method of claim 39, wherein the sensor device is implanted in aliving organism or in a culture system for a cell, tissue, or organ. 43.The method of claim 37, wherein the chemical agent is selected from thegroup consisting of H⁺, metal ions, glucose, bacteria, viruses, andorganic compounds.
 44. The method of claim 39, wherein the output signalis monitored over a period of time either continuously or at selectedtimes or time intervals.
 45. The method of claim 37, further comprising,prior to step (b): (a1) processing the liquid sample by filtration,purification, or adding one or more reagents to a liquid sample.
 46. Akit comprising the sensor device of claim 1, instructions for use, andoptionally one or more reagents used for processing a liquid sampleprior to flowing the liquid sample into said inlet port.
 47. A kitcomprising the sensor device of claim 14c and one or more reagentspreloaded into said one or more reservoirs.